Despite recent advances in research to develop a vaccine to slow or stop the progression of HIV-1 infection to AIDS, a fully or even partially efficacious vaccine remains elusive. There is a body of literature that provides circumstantial evidence suggesting that the Nef protein is a major determinant of HIV and SIV pathogenesis. Primates and humans infected with nef-deleted viral strains have delayed, or no pathogenicity. Mice and rats expressing nef as a transgene develop symptoms similar to those observed in human AIDS patients. Nef protein has been linked with the induction of apoptosis. Our preliminary results show: Soluble HIV-1, -2 and SIV Nef proteins, alone, induce apoptosis in CD4+ T lymphocytes. Nef proteins cytotoxic activity is mediated through the HIV-1 chemokine co-receptor CXCR4. The apoptotic motifs in the HIV-1 Nef protein have been localized in two 10-amino acid regions. The apoptotic region in SIV Nef protein has been localized to the C-terminal 20 amino acids. In tissue culture, Nef protein is shed into the extracellular environment. HIV-1 Nef protein has been identified in patient plasma samples, and shown to be apoptotic. A mouse model has been developed to study to study Nef-induced pathogenesis in vivo. Increased levels of apoptosis are observed in splenic lymphocytes in Nef-exposed mice. Nef and Pathogenicity.
The nef genes of Human Immunodeficiency (HIV) and Simian Immunodeficiency (SIV) Viruses encode 27-34 kd myristoylated proteins, which are expressed early after establishment of the provirus in host cells. Although largely dispensable for viral growth in cell culture, the expression of Nef results in higher viral loads and increased viral pathogenicity both in humans and in animal models for HIV/SIV infection. Correspondingly, nef-deleted SIV exhibits reduced pathogenicity, and lower viral loads when used to infect rhesus macaques. Interestingly, reversion to pathogenicity of SIVmacBK28-41 containing an attenuated nef gene was shown to be linked to the C-terminal region, which is rot found in HIV-1. Nef-deleted HIV-1 variants have been isolated from long-term survivors (LTS) of HIV-1 infection. A number of these LTS individuals remained healthy more than 10 years after a diagnosis of HIV-1 infection in the absence of ongoing antiretroviral therapy. For many of these patients, the lack of expression of Nef was the only observable attenuation of the HIV-1 virus.
Transgenic animal models have been developed to study effects of HIV expression on in vivo host systems. In these studies, lymphocyte depletion, as well as most of the organ system dysfunction found in AIDS patients was observed. More specifically, in mice transgenic for nef, lymphocyte depletion, as well as most of the tissue specific dysfunction found in AIDS patients is observed, and the nef-transgenic mice progress to death as seen in typical cases of HIV infected individuals.
Bystander Effect—Lymphocyte Depletion.
It has been proposed that reduction and killing of lymphocytes is directly linked to the viral load, and the depletion process is induced by viral infectivity. However, Anderson et al suggested that this model for pathogenesis made too many assumptions. They suggested that if one carefully analyzes the data and makes the minimum number of assumptions, viral load/direct consequences of infection do not explain the pathogenic effects of infection. It has been proposed that lymphocyte killing leading to depletion is a result of programmed cell death. Finkel et al. reported that apoptosis predominantly occurs in uninfected or bystander cells, with a distinct lack of cell killing in the productively infected cells themselves, (bystander effect). One relevant scenario to explain lymphocyte killing leading to depletion suggests that infected cells remain viable for the production and assembly of viral particles, while the surrounding uninfected CD4+ T lymphocytes are targeted for death. A number of studies have contributed to the “bystander effect” premise that the longevity of infected cells is due to intracellularly expressed Nef protein. It has been suggested that the virus uses a strategy by which intracellular Nef protects the HIV-1-infected host cell from pro-apoptotic signals through Nef-mediated interference with the function of the apoptosis signal-regulating kinase 1 (ASK1), while simultaneously promoting the killing of bystander cells through the induction of FasL. In these studies, it was shown that ASK1, which is a serine/threonine kinase, is essential for the complete signaling of Fas and TNF-alpha death-signaling pathways. However, in the presence of Nef, a complex consisting of ASK1 and Nef is formed which prevents the normal interaction of ASK1 with the death-signaling receptors. Other researchers report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both of these proteins for HIV-mediated up-regulation of FasL. Their data suggest that Nef can form a signaling complex with the T cell receptor (TCR) of T lymphocytes, thereby bypassing the requirement of antigen to initiate T cell activation and subsequent up-regulation of FasL expression. Long-term stable expression of Nef, which mimics persistent or latent infection in vivo, was observed to confer resistance against anti-Fas Ab-induced apoptosis through inhibition of caspase 3 and caspase 8 activation. HIV-1 Nef has also been suggested to augment HIV replication by prolonging the viability of infected cells through the blocking of p53-mediated apoptosis.
Nef and Apoptosis.
A second premise for the “bystander effect” scenario directly implicates viral proteins (i.e., Nef), or indirectly implicates virally stimulated cellular factors as mediators of bystander cell death. The N-terminal portion of the HIV-1 Nef protein has been shown to be involved in apoptosis of uninfected lymphocytes. Okada and colleagues have shown that soluble Nef protein, in conjunction with anti-Nef antibodies, is involved in the destruction of a broad spectrum of uninfected blood cells that includes CD4+ and CD8+ T-lymphocytes, B-lymphocytes, macrophages and neutrophils. This destruction leads to immune suppression. Adachi's laboratory reported that HIV-1 Nef, in concert with anti-Nef antibodies, induces apoptosis in CD4+ T lymphocytes. Specifically, Adachi's group claimed that CD4+ T cells are not killed when exposed to soluble HIV-1 Nef, but in the presence of cross-linking anti-Nef antibodies, Nef acquires cytotoxic activity against CD4+ T cells.
The bystander hypothesis hinges on the existence of soluble Nef protein in the extracellular environment. As might be expected because Nef is myristylated, it is closely associated with cellular plasmalemmas. It has been shown to (i) have multiple intracellular effects, (ii) be attached to the internal surface of the plasma membrane through the N-terminal myristylation group, and (iii) lose most of its important biological properties when the myristylation site is removed from nef by mutation. Between 60 and 200 molecules of Nef are incorporated in virions (where most are cleaved by the viral protease between residues 57 and 58 and associate with the viral core). It had been taken for granted that Nef protein does not have a significant extracellular presence. This is not correct. First, the cleaved, intravirion version of Nef could be released extracellularly through breakdown of normal or defective viral particles. Second, there is evidence that the full length Nef protein exists extracellularly in significant amounts. Full length myristylated Nef has now been shown to be released into the extracellular medium when expressed in mammalian cells in significant concentrations. Gould et al., showed that infected cells can release exosomes containing viral proteins. With the breakdown of these exosomes, the viral proteins would be released into the extracellular environment. Finally, soluble Nef protein has been detected in the sera of HIV-1 infected patients. These sera concentrations are more than enough to induce apoptosis in Jurkat cells. Thus, there is enough evidence of soluble, extracellular Nef to directly implicate Nef protein in bystander cell death leading to CD4+ T-cell depletion and AIDS.
Fujii et al, determined anti-Nef antibody titers in patient sera, and found that the anti-Nef antibody titers in these sera were inversely related to the Nef concentrations. They found that the five patient samples in which Nef was undetectable exhibited the highest titers of anti-Nef antibody. Earlier reports indicated that anti-Nef antibody levels in patient sera, both early in HIV-1 infection and after highly active antiretroviral therapy (HAART) vary significantly among individuals. Moreover, the relative strength of the anti-Nef response could be correlated with viral loads and disease progression. It was proposed that the lack of immune response to Nef may be correlated with a more rapid rate of progression to immune deficiency. Specifically, patients who developed a strong anti-Nef response at or near the time of infection were less likely to be rapid progressors to AIDS.